1. Field of the Invention
The present invention relates to a large-scale propagation and maintenance method of embryoid bodies generated from stem cells. More particularly, the present invention relates to a large-scale propagation and maintenance method of embryoid bodies retaining their intrinsic characteristics for a long period of time, comprising the step of continuously subculturing embryoid bodies that are primarily generated from embryonic stem cells or from induced pluripotent stem cells, an EBD for dividing embryoid body, and a culture vessel for culturing embryoid bodies.
2. Description of the Related Art
Stem cells have a strong self-renewal capacity that enables these cells to regenerate damaged tissue, and thus have been recently recognized as an important biological source in regenerative medicine and medical technology development. Stem cell-based technologies are expected to create a highly valuable new industry subset in the future medical industry, and are expected to have tremendous impact on other industrial fields. In particular, differentiation-induction technology used to obtain functionally superior tissue-specific differentiated cells from stem cells is recognized as a core technology likely to greatly expand the future stem cell industry.
Pluripotent human embryonic stem cells (hESC) retain an ability to differentiate into any cell type present in the body, and thus are regarded as an attractive cell source for obtaining differentiated cells. Technology to obtain differentiated cells from hESC has been competitively developed worldwide. Tissue-specific differentiated cells from human stem cells provide great opportunities for basic research in human development, cell therapy, and the testing of the efficacy and cytotoxicity of candidate drugs. The demand for tissue-specific differentiated cells is continuously increasing.
Since a de-differentiation strategy using de-differentiation transcription factors was successfully developed by Dr. Yamanaka in Japan, 2006 (Takahashi et al, Cell, 2006), many research groups have conducted investigations into human induced pluripotent stem cells (iPSC), that is, pluripotent stem cells similar to human embryonic stem cells that can be generated from human somatic cells (Takahashi et al, Cell, 2007; Yu et al, Science, 2007; Park et al, Cell, 2008). As successful development of de-differentiation strategy makes it possible to generate pluripotent stem cells from patient somatic cells in a comparatively easy way, utilization of patient-derived iPSC for development of basic and applied technology will rapidly grow. Tissue-specific differentiated cells from human stem cells provide great opportunities for basic research in human development, cell therapy, and testing of the efficacy and cytotoxicity of candidate drugs. The demand for tissue-specific differentiated cells is continuously increasing.
The differentiation-induction technique for the maintenance and proliferation of undifferentiated hESCs and hiPSCs involves varied and time-consuming methods for increasing the number of cells, which is a major drawback to the development of related techniques. Currently, most protocols for generating differentiated cells from hESCs and hiPSCs proceed by way of the formation of embryoid bodies. That is, in vitro tissue-specific differentiation of hESCs and hiPSCs requires an initial spontaneous formation of embryoid bodies (EBs) in suspension cultures, which is a common and critical intermediate for the induction of lineage-specific differentiation. The conventional methods of forming embryoid bodies have limitations in that the production yield considerably depends on the number of the starting stem cells, and the formed embryoid bodies are heterogeneous in size and number. For this reason, current studies have been made to develop a formation and culturing system of human embryoid bodies using multiwall and microfabrication techniques as well as stirred and mixed suspension culture systems (Cameron, C. M. et al., Biotechnol Bioeng 94, 938-948, 2006; Gerecht-Nir, S. et al., Biotechnol Bioeng 86, 493-502, 2004; Moeller, H. C. et al., Biomaterials 29, 752-763, 2008; Torisawa, Y. S. et al., Lab Chip 7, 770-776, 2007). However, current differentiation techniques using embryoid bodies absolutely depend on the supply of qualified hESCs or hiPSCs in terms of both quantity and quality. For differentiation induction, healthy, qualified, undifferentiated hESCs or hiPSCs should be obtained at first, and embryoid bodies are formed and produced in 1:1 correspondence. The number of embryoid bodies increases for a predetermined period (approximately 2 weeks), but tends to decrease thereafter, and thus there is a need to obtain a large number of undifferentiated hESCs or hiPSCs at the starting point, in order to increase the number of differentiated cells. That is, a major bottleneck in efficient differentiation is a failure to acquire a large number of undifferentiated hESCs or hiPSCs, which require varied, time-consuming methods.
Accordingly, the present inventors have made many efforts to develop a method capable of mass-producing embryoid bodies which are a mother of differentiated cells, while reducing dependency on the use of undifferentiated pluripotent stem cells. As a result, they have established a method for continuously culturing embryoid bodies through a simple subculture process. During this process, they found that the method allows for the long-term maintenance and culture (for over 20 continuous passages) of EB-constituting cell populations without loss of their survival, proliferation, and differentiation (into three germ layers). Further, micropatterning techniques have been developed for the improvement of differentiation from embryoid bodies, but the large scale-production of the initial material of undifferentiated hESCs and hiPSCs is essential for the scale-up propagation of embryoid bodies in the current techniques. That is, since the current techniques absolutely depend on the availability of undifferentiated hESCs and hiPSCs, there has been no progression in the improvement of large-scale production systems of embryoid bodies. Automatic systems have been developed to improve the culture conditions of embryoid bodies. These systems contribute to effective maintenance of embryoid bodies once formed for a predetermined period of time, but do not satisfy the demand for large-scale production thereof. In order to solve this problem, the present inventors have developed a method for large-scale production of embryoid bodies by uniform dividing and subculturing, thereby completing the present invention.